A study of the collagen-binding domain of a 116-kDa Clostridium histolyticum collagenase.
The Clostridium histolyticum 116-kDa collagenase consists of four segments, S1, S2a, S2b, and S3. A 98-kDa gelatinase, which can degrade denatured but not native collagen, lacks the C-terminal fragment containing a part of S2b and S3. In this paper we have investigated the function of the C-terminal segments using recombinant proteins. Full-length collagenase degraded both native type I collagen and a synthetic substrate, Pz-peptide, while an 88-kDa protein containing only S1 and S2a (S1S2a) degraded only Pz-peptide. Unlike the full-length enzyme, S1S2a did not bind to insoluble type I collagen. To determine the molecular determinant of collagen binding activity, various C-terminal regions were fused to the C terminus of glutathione S-transferase. S3 as well as S2bS3 conferred collagen binding. However, a glutathione S-transferase fusion protein with a region shorter than S3 exhibited reduced collagen binding activity. S3 liberated from the fusion protein also showed collagen binding activity, but not S2aS2b or S2b. S1 had 100% of the Pz-peptidase activity but only 5% of the collagenolytic activity of the full-length collagenase. These results indicate that S1 and S3 are the catalytic and binding domains, respectively, and that S2a and S2b form an interdomain structure.
Matsushita
O
O
Department of Microbiology, Faculty of Medicine, Kagawa Medical University, Miki-cho, Kita-gun, Kagawa 761-0793, Japan.
Jung
C M
CM
Minami
J
J
Katayama
S
S
Nishi
N
N
Okabe
A
A
D002384:Catalysis; D003013:Clostridium / Q000201:enzymology*; D003094:Collagen / Q000378:metabolism*; D017364:Collagenases / Q000235:genetics / Q000378:metabolism*; D018093:Gelatinases / Q000378:metabolism; D005982:Glutathione Transferase / Q000378:metabolism; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017384:Sequence Deletion; D013379:Substrate Specificity
D011993:Recombinant Fusion Proteins; D003094:Collagen; D005982:Glutathione Transferase; D017364:Collagenases; D018093:Gelatinases